The function of a number of proteins remains elusive, which slows down understanding of fundamental aspects of cell mechanisms and impedes advance in diagnostic and treatment of many diseases and in personalized medicine. One of the main obstacles to better understand a given protein's functions is that proteins generally interact with many other partners (proteins, nucleic acids, sugars, lipids . . . ) and many of these interactions are weak and transient, albeit of critical importance.
A series of methods have been developed to provide a better view on protein interaction (Lowder et al., Visualizing protein partnerships in living cells and organisms. Curr Opin Chem Biol 15, 781-788, 2011), among them immuno-precipitation assays, two-hybrid systems and variants like substrate complementation systems, Fluorescence Energy Transfer (FRET) or related techniques.
A typical procedure adopted by scientists is to take advantage of the high throughput capability of the two hybrid system, generally in yeast, to obtain an exhaustive list of potential partners for a protein of interest and then to control whether the proposed interactions are also detected with one of the two other methods (immuno-precipitation assays or FRET).
Each of these methods suffers from numerous drawbacks. Immuno-precipitation assays require cell lysis (Paul et al., Analyzing protein-protein interactions by quantitative mass spectrometry. Methods 54, 387-395, 2011), specific antibodies and adsorption onto non physiological substrates leading to false positives and false negatives. In addition, information obtained does not reflect necessary what may happen in living cells and most critically these methods detect strong interactions, even though some improvements have been done on this point (Lee et al., Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions. Nat Protoc 8, 2045-2060, 2013) (Jain et al., Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484-488, 2011). Two-hybrid systems can detect protein interactions in living cells but require two or more chimeric proteins and are unable to monitor rapid biochemical events taking place in the cells (Lievens et al., Mammalian two-hybrids come of age. Trends Biochem Sci 34, 579-588, 2009). In addition, weak and transient interactions are not detected. FRET can also detect protein interactions in living cells but the FRET signal is strongly dependent on the distance and the orientation of the donor and acceptor labels placed on the bait and target proteins, respectively. The detection of weak interactions is also challenging due to the low signal to noise ratio (Lam et al., Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005-1012, 2012). In addition, when the bait and prey interact with each other but the donor is well separated from the acceptor (>4-5 nm), the FRET signal is weak.
Engineered fluorescent proteins suitable for FRET are further taught in WO 2013087921A1, such as pairs of donor and acceptor fluorescent proteins. Other alternatives to the yeast two-hybrid system, but distinct from FRET, have been developed for eukaryotic cells.
U.S. Pat. No. 7,244,614B2 teaches recombinant fusion proteins for detecting the binding of a molecule of interest, comprising a detection domain, a binding domain, and a localization domain such as a Nuclear Localization Signal (NLS) or a Nuclear Export Signal (NES).
WO 2000017221A1 relates to a method for measuring protein-protein interactions in living cells, using a first heterologous conjugate with a detectable group and a second heterologous conjugate that binds to an internal structure within said cells. According to WO 2000017221A1, an <<internal structure>> includes any non-uniformly distributed cellular component, including for instance proteins, lipids, carbohydrates, nucleic acids and derivatives thereof.
However there remains a need for novel methods and tools suitable for detecting weak and transient interactions between ligands in living cells.
There also remains a need for such methods which remain, at the same time, suitable for high-throughput screening.
There also remains a need for methods which provide suitable and physiological conditions in the context of a living cell, and which also remain suitable for the detection of ligand interactions in real-time.